T cell immunity against early antigens of Human
Papillomavirus type 16
Jong, Annemieke de
Citation
Jong, A. de. (2005, January 27). T cell immunity against early antigens of
Human Papillomavirus type 16. Retrieved from
https://hdl.handle.net/1887/624
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Corrected Publisher’s Version
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of Leiden
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T CELL IMMUNITY
AGAINST EARLY ANTIGENS
OF HUMAN PAPILLOMAVIRUS
TYPE 16
T CELL IMMUNITY
AGAINST EARLY ANTIGENS
OF HUMAN PAPILLOMAVIRUS
TYPE 16
•
t c ell immuni t y
ag a ins t e a rly a n t igens
of hum a n pa pil l om av irus t y pe 16
•
Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van de Rector Magnifi cus Dr. D.D. Breimer,
hoogleraar in de faculteit der Wiskunde en
Natuurwetenschappen en die der Geneeskunde,
volgens besluit van het College voor Promoties
te verdedigen op donderdag 27 januari 2005
promotiecommissie promotores Prof. dr. C.J.M. Melief
Prof. dr. P. Vermeij co-promotor Dr. S.H. van der Burg
referent Prof. dr. M.A. Stanley, University of Cambridge, UK overige leden Prof. dr. C.J.L.M. Meijer, VUMC, Amsterdam
Prof. dr. J.H.F. Falkenburg Prof. dr. G.J. Fleuren Dr. R. Offringa Dr. G.G. Kenter
The studies described in this thesis were performed in the departments of Immunohematology and Blood Transfusion, Clinical Pharmacy and Toxicology, Gynecology, and Pathology of Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
© A. de Jong, 2004.
Niets uit deze uitgave mag worden verveelvoudigd en/of openbaar gemaakt zonder voorafgaande schriftelijke toestemming van de auteur. No Part of this thesis may be reproduced in any form without written permission from the author.
isbn 90-901898 1-5
Foto’s omslag Hinomaru, Miyashita Ryosuke ( ), with kind permission; Cryoelectron micrograph of HPV particle, T. Baker, with kind permission. Vormgeving Sam Gobin, Leiden
Druk Offsetdrukkerij Nautilus, Leiden
The mind is like a parachute;
it only works when it’s open
– O. Welles
contents
1
Introduction — 9
2
Frequent detection of HPV 16 E2-specifi c T-helper immunity in healthy
subjects — 25
3
Frequent display of HPV 16 E6-specifi c memory T-helper cells in the
healthy population as witness of previous viral encounter — 4 1
4
HPV 16-positive cervical cancer is associated with impaired CD4+ T cell
immunity against early antigens E2 and E6 — 53
5
IL- 10 secretion characterizes HPV-specifi c CD4+ T cell repertoire in
cer-vical tumor and tumor-draining lymph nodes — 67
6
Enhancement of HPV 16 E6 and E7-specifi c T cell immunity in healthy
volunteers through vaccination with TA-CIN, an HPV 16 L2E7E6 fusion
protein vaccine — 83
7
Rapid enrichment of HPV-specifi c polyclonal T cell populations for
adoptive immunotherapy of cervical cancer — 97
8
General Discussion — 1 1 1
9
Samenvatting — 12 1
10
Curriculum vitæ — 125
Human papillomaviruses (HPV) underlie the carcinogenesis of a substantial fraction of human can-cers, including cancer of the uterine cervix. Cervical cancer is the second leading cause of cancer-related deaths among women worldwide, and it is the fi rst malignancy acknowledged by the World Health Organisation to be virally induced in essentially all cases [ 1-3]. The close association between an infectious agent and cancer provides an important opportunity to target this disease by means of immunotherapeutic strategies. The fact that anogenital HPV infections are extremely common implies that HPV infection itself is not the decisive factor in cancer development. Rather, it is the failure to control HPV infection and the subsequent establishment of persistent infection that pre-disposes progression towards malignancy [4, 5]. This failure of immunosurveillance is observed only in a minority of infected subjects, whereas most infected individuals fortunately clear the infection without the development of HPV induced lesions. Clues as to how lesions expressing viral antigens can progress in the face of immune defence are likely to be found in the immune response against HPV antigens. Detailed knowledge of this HPV-specifi c immunity, both in individuals successfully controlling HPV infection and those who have evidently failed to do so, will assist the rational design of therapeutic interventions in the course of HPV induced disease.
Human Papillomaviruses
Human papillomaviruses are small double-stranded DNA viruses, and over a hundred types have been identifi ed and fully or partially sequenced to date [6]. All identifi ed types are strictly epithelio-tropic, and depend on the differentiation process of the external epithelium for viral propagation. Papillomaviruses infect the epithelial cells either of the skin or of the anogenital and oropharyngeal mucosa. Based on their oncogenic potential, human papillomaviruses can be divided into low-risk types, causing benign epithelial proliferations, and high-risk types, which can incidentally result in malignant transformation of epithelial cells [7]. HPV type 16 (HPV 16) is the most prevalent high-risk type in HPV-associated anogenital malignancies – HPV 16 DNA is present in more than 50 % of cervi-cal tumors [8] – and forms the focus of the HPV-specifi c immunity outlined in this thesis.
HPV 16 viral particles contain the double-stranded closed circular DNA genome, associated with histone-like proteins and encapsidated by 72 capsomers. The HPV 16 genome consists of approxi-mately 7900 base pairs harbouring 8 open reading frames (Figure 1). The genome can be divided into three regions: a non-coding long control region (LCR), an early (E) and a late (L) region. The late genes encode structural proteins, whereas the early genes mainly encode proteins with regulatory functions engaged in genome persistence and DNA replication. The LCR harbors specifi c enhancer elements, which are responsive to both viral and cellular regulatory factors [9]. The functions of the HPV 16 encoded proteins are described in Table 1.
Viral life cycle
The papillomavirus life cycle differs from all other virus families by its tight restriction to the differenti-ating epithelium. Establishment of infection requires the availability of epidermal or mucosal epithe-lial cells that are capable of proliferation (basal cells). After infection of the basal stem cell, the virus utilises the host DNA replication machinery to multiply a few of its viral genomes [9]. In the basal cell, HPV genomes are established as episomes at low copy number, which replicate in synchrony with the cellular DNA replication. The viral gene expression is largely suppressed in the basal layer, although the limited expression of specifi c early viral proteins (e.g. E5,E6,E7) results in enhanced proliferation of the infected cells and their lateral expansion [6, 25]. The restricted expression of viral genes in the basal cells is suggested to be the result of tight control by cellular factors [26, 27].
The episomal HPV genomes persist in the nucleus of the infected stem cells and are distributed during mitosis on to the daughter cells. Therefore, the infected stem cell acts as a reservoir. In
con-Table 1. HPV 16 protein function
Protein Function
E 1 DNA helicase. Essential for initiation of viral replication [ 10]
E2 Interacts withs E 1, facilitating the binding of E 1 to origin of replication [ 1 1]
Viral transcription factor [ 12, 13]
E4 Associates with keratin cytoskeleton of epithelial cells resulting in collapse of cytokeratin network facilitating
viral particle release [ 14]
E5 Interferes with endocytic traffi cking, reducing IFNg-induced HLA class II surface expression [ 15, 16]
Stimulates cell growth by enhancing effect of growth factors [ 17]
Prevents apoptosis following DNA damage [ 18]
E6 Prevents apoptosis by targeting the degradation of p53 and Bak [ 19, 20]
Activation of telomerase [2 1]
E7 Prevents cell-growth arrest / differentiation by the release of E2F transcription factor from pRB-E2F complex
(E7 binds to pRB and pRB family members), resulting in deregulation of G 1 / S cell cycle checkpoint [22]
Blocks function of cyclin-dependent kinases p2 1 and p27 [23, 24]
L 1 Major capsid protein
trast to uninfected keratinocytes, which exit the cell cycle as soon as they detach from the basement membrane, HPV infected cells enter into the S phase after reaching the suprabasal layer. This entry into the S phase results in amplifi cation of the viral genomes to thousands of copies per cell [9]. Papillomaviruses herewith possess a mechanism to overcome the block in DNA synthesis that occurs along with the differentiation of the epithelial cells, although this does not result in full replication of the genome of the differentiated host cells [28]. ‘Late’ viral gene expression is initiated in the upper layers of the epithelium, where the viral particles are assembled and released (Figure 2). The virus actually delays nuclear condensation in differentiating keratinocytes by halting apoptosis until viral replication is completed [29].
The development of cervical cancer
Persistence of high-risk cervical HPV infection is characterized by the formation of dysplastic precur-sor lesions, which can be classifi ed cytologically (Pap 1-5) or histologically (CIN I-III). The histologi-cal features of more advanced dysplastic lesions include a signifi cant extension of actively replicat-ing cells into the upper parts of the epithelium, loss of coordinated epithelial differentiation, and together with this loss of the complete viral life cycle. It has been proposed that CIN lesions constitute a progressive neoplastic disease continuum leading up to invasive cervical carcinoma. However, the majority of low-grade lesions (CIN I/II) will regress spontaneously indicating that the development of CIN and cervical carcinoma is not a one-way process [30, 3 1]. Furthermore, the disease process can show considerable variation; low-grade lesions can persist for years in some patients, whilst in others immediate progression to high-grade dysplasia (CIN III) is observed [32, 33]. So far, treatment of HPV-induced high-grade lesions and cancer is mostly limited to surgical removal, which has proven suc-cessful in those cases in which the lesions are locally confi ned. Because this treatment modality is symptomatic and lacks an anti-viral component, there is a risk of recurrent disease [34-36].
The role of HPV (onco)proteins in malignant transformation
Persistence of high-risk HPV infection, causing genomic instability is a prerequisite for disease progression and the development of precursor lesions. However, malignant transformation of the infected keratinocytes requires an additional step, involving the integration of HPV DNA into the host genome. During this process of viral integration, a substantial part of the genome is frequently deleted [37, 38]. In the resultant organization of HPV DNA, the viral transcripts spanning the E6 and E7 region are often linked to fl anking cellular sequences, and subsequently the transcription
E6 E7 E1 E2 E4 E5 L1 L2
HPV16
LCRFigure 1. HPV 16 genome. Schematic representation of the circular HPV genome. The open reading frames (ORFs) are depicted
of the oncogenes can be modulated (enhanced) by fl anking host-cell promoters [39]. In addition, co-transcribed cellular sequences may result in increased stability of the mRNA encoding the viral oncoproteins [40]. Overall, the integration of high-risk HPV into the host genome results the consti-tutive expression of the viral oncogenes.
The E2 protein is described to be the major regulator of viral protein expression, and capable of suppressing E6 and E7 oncogene expression [4 1]. Therefore, the deletion of the E2 gene in the process of viral integration has been held responsible for the unleashed expression of both oncoproteins. This has, however, been contradicted by a recent study in which the suppressive effect of E2 on E6 and E7 expression was observed only for integrated and not for episomal viral DNA [42]. Therefore, the increased oncoprotein expression after viral integration may more likely be explained by factors other than the absence of the controlling function of E2, such as the aforementioned enhanced transcrip-tion of the oncogenes by fl anking host-cell promoters, and the increased stability of chimeric tran-scripts. Although the E 1 and/or E2 genes are often disrupted and part of the viral genome deleted in HPV 16 positive cervical carcinoma, episomal copies of the viral DNA can often still be detected in the tumor cells, resulting in a variable expression of the HPV 16 proteins. However, the lack of cellular differentiation at this stage of disease precludes completion of the viral life cycle.
The oncoproteins E6 and E7 proteins play a signifi cant role in the process of malignant transformation. Independently they have the capacity to immortalize various human cell types in vitro; the effi -ciency of which is increased when the oncoproteins are expressed together [43]. A number of interac-tions have been reported between high-risk E6/E7 and host-cell proteins (Figure 3). For E6, the most prominent functions originate from its interaction with, followed by the degradation of, p53 and the pro-apoptotic protein Bak. These events result in resistance to apoptosis and an increase in chromo-somal instability. In addition, E6 has been shown to associate with myc proteins in vivo, resulting in the cooperative activation of the telomerase reverse transcriptase promoter (references in Table 1). E7 interacts with the RB protein, which releases the transcription factor E2F from RB inhibition.
Figure 2. HPV infection and propagation. HPV infection of the cervix often occurs at the so-called squamocolumnar junction or transformation zone. This is where the columnar epithelium of the endocervix is connected to squamous epithelium of the exocervix, and the undifferentiated basal stem cell is readily accessible for HPV infection. After entry of the HPV virion into the basal epithelial (stem)cell, the viral genome is amplifi ed to several copies. The infected cell divides along the basement membrane and then matures vertically. In the suprabasal layers of the epithelium HPV early proteins are abundantly expressed and the viral replication takes place. Several of these proteins (e.g. E6 and E7) interfere with epithelial differentiation. Only in the most superfi cial layers of the epithelium, E4 and the late viral genes L 1 and L2 and the E4 re-expressed, and the HPV DNA is encapsidated and the virions are released at the epithelial surface.
E4 L2 L1 E6 E7 E1 E2 E5 HPV virion Viral episome
The released E2F functions as a transcriptional activator, resulting in deregulated G 1/S transition. Moreover, E7 is shown to block the function of the cyclin-dependent kinase inhibitors p2 1 and p27. High E2F activity might lead to apoptosis in E7-expressing cells, however, E6 in turn prevents this by guiding the degradation of apoptosis-inducing proteins p53 and Bak (references in Table 1). Taken together, the interference of E6 and E7 with cell cycle control, allows the accumulation of mutations in host cell DNA, thereby promoting malignant cell transformation.
Immune responses against hpv
Immune evasion and regulation in the HPV-infected epithelium
Anogenital HPV infections are extremely common, and the cumulative lifetime incidence is esti-mated to be as high as 80-85 % [44]. The fact that most infections are cleared and that low-grade CIN lesions often regress spontaneously, indicates that in the majority of individuals the immune system succeeds in eliminating the virus before malignant disease can develop. Indirect evidence for the major role of the cellular immune response in this process is given by the fact that the prevalence of persistent HPV infections and HPV-positive lesions is greatly increased in immunosuppressed sub-jects, such as transplant recipients and HIV-positive patients [45-48].
The duration of a transient anogenital HPV infection ranges from 7– 14 months [32, 49], indicat-ing that the virus is capable of persistindicat-ing in the host for some time before elimination. This lag time between HPV infection and clearance is at least partially linked to the evasion of and/or interference with innate immune defences [50]. Lack of innate immune triggering will in turn avoid activation of the adaptive immune system to eradicate virus-infected cells. Several characteristics of the viral infection contribute to the temporary circumvention of immune triggering, including the minimal disturbance of the epithelial architecture initially caused by the virus and the absence of signifi cant keratinocyte lysis and infl ammation. Furthermore, the differentiation dependent protein expression limits abundant production of viral proteins to the top epithelial layer, less accessible and visible to
Transcriptional activation inactive complex Transcriptional repression pRB E2F E7 E2F E7 pRB G1 S G2 M E6-AP E6 E6-AP E6 E6-AP E6 p53 E6-AP E6 p53 Ub Ub Ub Ub Ub Ub Proteasomal degradation E6 Activation of telomerase Inactivation of Bak
Synergy of E6 and E7 resulting in: – Cell proliferation – Prevention of apoptosis – Immortalization – Malignant transformation p53 Bak Apoptosis Degradation of p53
Figure 3. Mechanisms of E6 and E7 interference with cell cycle control. Together with E6-AP, the E6 protein
cells of the immune system. Moreover, the uptake of HPV 16 virus-like particles (VLP) by Langerhans cells – the antigen presenting cells (APC) present in the epithelium – does not result in activation of these cells [5 1]. This in contrast to the uptake of VLP by monocyte-derived dendritic cells (DC), which does induce the upregulation of maturation markers on the DC and the secretion of IL- 12. Taken together, the overall lack of infl ammatory signals involved in papillomavirus infection allows a temporary evasion of immune surveillance.
Besides the avoidance of immune system activation, papillomaviruses also harbour mechanisms to actively interfere with innate immune triggering. The oncoproteins E6 and E7 have been shown to prevent the immunoregulatory effects of the type I interferon pathway by physically interfering with specifi c components of this pathway, e.g. inhibition of Interferon regulatory factors IRF 1 and IRF3 (reviewed in [52]). Furthermore, the selective downregulation of MCP- 1 expression by E6 and E7 in epithelial cells may also contribute to the program of HPV immune evasion, as this chemokine is particularly relevant in the setting of viral infection due to its ability to attract monocytes, memory T cells and NK cells in vivo [53]. Interference with the initiation of an adaptive immune response has been suggested by Matthews et al., who demonstrated E6-mediated downregulation of E-cadherin, resulting in Langerhans cell (LC) depletion of HPV 16-infected epithelium [54]. This may limit the pre-sentation of viral antigens by LC, thus preventing the initiation of a cell-mediated immune response and promoting survival of the virus.
Although keratinocytes have the capacity to secrete pro-infl ammatory cytokines under certain conditions, they can also produce immunoregulatory factors such as TGFb and IL- 10. Both these cytokines play an essential role in the differentiation of Langerhans cells (LC) and their retention in the epidermis [55, 56]. Furthermore, these factors can inhibit the induction of LC maturation by pro-infl ammatory factors. It is evident that LC-mediated activation of T cells in the regional lymph nodes requires the pro-infl ammatory signals to dominate over the homeostasis of LC as controlled by TGFb and IL- 10. Lack of suffi cient pro-infl ammatory signals will result in immunological ignorance or toler-ance [57]. Several reports describe increased levels of IL- 10 in HPV-induced high-grade CIN lesions [58-60], and an increase in this immunoregulatory cytokine may have profound effects both on the resident LC in the skin, infl uencing their migration pattern and their antigen presenting capac-ity after migration to draining lymph nodes [6 1]. Furthermore, local IL- 10 secretion in the HPV-induced lesion may negatively affect the functionality of many types of effector cells migrating to the lesions [62]. Giannini et al. suggested that immunosurveillance within the epithelium of the transformation zone – compared to the exocervix – is intrinsically perturbed by the altered expres-sion of chemokines/cytokines (e.g. TNF-a, MIP3a) and by the concomitant diminished density of immature LC. Furthermore, the allo-stimulatory capacity of LC derived from the transformation zone was reduced compared to those from the exocervix, and the function of high-grade CIN-derived LC appeared even further incapacitated [63].
Notwithstanding these considerations, the situation during natural HPV infection appears to gen-erally favor the induction of effective immunity rather than tolerance, as indicated by the fact that the vast majority of active HPV infections are eventually eliminated. However, in susceptible indi-viduals, a period of decreased vigilance of the immune system can result in minor disturbance of the balance between pro- and anti-infl ammatory signals and thereby allow the virus suffi cient time to establish a status quo in which HPV infected and/or HPV-transformed cells are diffi cult to elimi-nate. The fact that – in contrast to low-grade CIN lesions – more advanced cervical lesions rarely show spontaneous regression, underlines the need for the immune system to act during the early pre-malignant phase in order to be effective against the virus.
activation of dendritic cells (DC) or LC in the presence of these cytokines was shown to result in APC with decreased costimulatory capacity, lacking IL- 12 secretion, which are poor stimulators of Th 1/CTL immunity and which can even induce T cell tolerance [69-73]. In several cases such APC were shown to rather induce Th2-type immunity [74, 75], which is suggested to be relatively ineffective against solid tumors and which seems to prevail in patients with progressed cancers [64, 76].
Viral latency
Many lines of evidence point towards the existence of a latent phase of papillomavirus infection, which can be defi ned as the presence of viral DNA in the absence of differentiation-dependent virion production [77]. Indications for viral latency are mainly derived from both the Canine oral papil-lomavirus (COPV) and Cottontail rabbit papilpapil-lomavirus (CRPV) model by the detection of viral DNA in post-regression tissue [78] and the induction of viral protein expression in previously infected sites by UV irradiation [79]. In humans, HPV latency has been suggested in the larynx and trachea of patients with recurrent respiratory papillomatosis (RRP). HPV DNA could be detected in biopsies from clinically normal laryngeal and tracheal tissues derived from RRP patients [80]. Indirect evi-dence for a latent phase in HPV infection is given by the rapid appearance of multiple HPV lesions in immunocompromised patients [77]. Most studies do not allow distinction between true latency and subclinical infection. In the immunocompetent host, HPV infection may be held in a subclinical state by effective cellular immunity, which can then readily evolve under immunosuppressive conditions. Mechanism of viral clearance
The term clearance is used to indicate the absence of active viral infection; regarding the presumed viral latency phase, this does not indicate a complete eradication of viral DNA. Little is known of the clearance process of HPV infections and HPV-induced lesions in the female genital tract. The regres-sion of genital warts – associated with low-risk HPV infection of the external genital mucosa – is shown to be characterized by an active cell-mediated immune response [8 1]. Regressing warts con-tained signifi cantly more T cells and macrophages than did non-regressing controls, and in this regression process, CD4+ T cells predominated in both the wart stroma and the surface epithelium. The great importance of CD4+ T cells in the control of HPV infections, is substantiated by reports showing that low CD4+ T cell count is strongly associated with multiple HPV infections, high viral load, and viral persistence in HIV-infected individuals and by the extensive HPV induced lesions observed in HIV negative patients with idiopathic CD4+ T-lymphocytopenia [47, 82-84].
Cellular immunity against HPV proteins
The confi nement of HPV infection to the epithelium renders the Langerhans cell (LC) the profes-sional APC primarily responsible for the induction of T cell immunity against HPV antigens. Impor-tantly, as the HPV infection cycle is specifi cally adapted to the keratinocyte differentiation program, it is unlikely that HPV infection of LC will result in the expression of the HPV antigens by the LC themselves. Therefore, the priming of T cell immunity against HPV will depend on the uptake and (cross)presentation of HPV antigens by the antigen presenting cells. The effi ciency of this process will be affected by the levels to which the viral proteins accumulate in the infected keratinocytes that serve as antigen source and by the release of HPV proteins from apoptotic/(necrotic) keratinocytes. Effective intervention of the cellular immune system in productive HPV infection is likely to ben-efi t from T cell immunity against papillomavirus immediate early antigens, because these antigens are expressed throughout suprabasal layers of the infected epithelium [9]. Although T cells directed against ‘late’ HPV antigens can defi nitely play a role in the HPV immune response, these anti-gens are only expressed when the viral infection cycle is almost complete and the differentiating keratinocyte has moved up in the epithelium [89]. Therefore, cellular immune responses against these antigens will most likely not result in effective viral clearance. Studies addressing T cell immu-nity against HPV have focused primarily on type 16 (HPV 16) because of the high prevalence of this oncogenic type high-grade CIN lesions and cervical carcinoma. More specifi cally, responses against the E6 and E7 oncoproteins of HPV 16 have been extensively studied, due to their constitutive expres-sion in HPV 16-positive (pre)malignant leexpres-sions. However, during productive viral infection and in low-grade CIN, other early proteins (E 1, E2 and E5) are also widely expressed in the (supra)basal epithe-lium and may also provide targets for the cellular immune system [90, 9 1].
So far, no clear-cut association has been established between HPV-specifi c T cell responses and protection against progression of HPV-induced disease. In a cross-sectional analysis by de Gruijl et al. it was shown that strong Th responses against HPV 16E7, as determined by cytokine-induced prolifera-tion of a reporter cell line, were associated with persistence and progression of HPV 16-positive lesions. However, a longitudinal analysis by the same group showed that HPV 16E7-specifi c Th responses were associated with both clearance and persistence, in which it was suggested that these responses devel-oped as a consequence of increased antigen availability resulting from either clearance of progression of cervical lesions [92, 93]. Our previous cross-sectional analysis of HPV 16E7 responses by IFNg ELISPOT in healthy individuals and patients with HPV 16-positive cervical cancer or high-grade CIN showed fre-quent E7-specifi c responses in HPV 16+ patients, whereas these were rarely detected in healthy controls [94]. In a longitudinal study performed by Kadish et al. proliferative responses against HPV 16E7 and/or E6 in CIN patients were found to be associated with clearance of HPV infection and regression of CIN [95]. It must be noted, however, that only a fraction of the CIN patients participating in this study was HPV 16 positive, and only the C-terminal part of E7 was used as a readout. Several other studies report highly variable percentages of HPV 16E7- and/or E6-specifi c Th-responses in patients [96, 97]. Like in the case of Th responses, contradictory results have been obtained regarding CTL responses against the HPV 16-derived oncoproteins. Lack of E6-specifi c CTL was found to be an important factor in HPV 16 persistence [98], whereas a study by Bontkes et al suggested that CTL against E6 and E7 are generally only found in persistent CIN [99]. In concordance with the latter study, Ressing et al. detected CTL responses against the – previously determined – HLA-A*020 1 restricted epitope HPV 16E7 1 1-20 in cervical carcinoma and CIN patients (HPV 16- and HLA-A*020 1-positive) and not in
healthy individuals [ 100]. Youde et al. showed by HLA-A*020 1 tetramer staining that – after a short in vitro stimulation – the frequency of CD8+ T cells specifi c for E7 1 1-20 was signifi cantly higher in
HPV 16+ CIN III patients than in healthy controls [ 10 1].
are HPV 16 positive) and, more importantly, by the differences in technical approach. Several older studies involve multiple in vitro stimulations, and do therefore not truly refl ect the in vivo situation. Furthermore, the choice of antigen – recombinant protein or synthetic peptides spanning only a lim-ited region of the total protein – has most likely infl uenced the outcome of the analyses.
A limited number of studies has addressed the cellular immunity against other HPV antigens. Bontkes et al. studied the HPV 16E2-specifi c Th-response in CIN patients and observed that Th cell responses against the C-terminal domain of E2 frequently occurred at the time of HPV clearance, although no signifi cant association between E2-specifi c Th responses and disease outcome was observed [ 102]. Proliferative responses against the E5 protein of HPV 16 were signifi cantly reduced in the patients with high-grade CIN and cervical carcinoma, as compared to patients with low-grade lesions [ 103]. The responses were exclusive to those with HPV 16-positive lesions, but the difference in response frequency lost statistical signifi cance when only the HPV 16-positive patients were included in the analysis. Therefore, the observed results require confi rmation in a larger HPV 16-positive popu-lation. T cell responses against the E4 antigen of HPV 16 have shown no correlation with infection and/or disease [ 104]. As for the late antigens L 1 and L2, expression of E4 is restricted to the upper differentiated layers of the epithelium [89], and as such T cell responses against this antigen are not likely to contribute signifi cantly to viral clearance. The late antigen HPV 16L 1 is a major target of the cellular immune system, as L 1-specifi c responses were abundantly detected in the peripheral blood of HPV 16+ CIN patients [ 105, 106]. The analyses were not performed with the entire L 1 antigen, but only with a predetermined immunogenic region. Again, these T cell responses were not associated with disease severity, as they were observed both in those with virus clearance and in those with per-sistence, irrespective of CIN grade. The high degree of L 1 conservation between HPV types, brings up the question whether L 1 cross-reactive T cells – recognizing the HPV 16L 1 peptides in vitro – have con-tributed to the high percentage of responders. Overall, these studies that addressed HPV 16-specifi c cellular immunity indicate that responses against the viral proteins are occasionally induced at one point during HPV 16-induced disease, but their role in protection against disease remains unclear. Humoral immune responses against HPV
Serum IgG antibodies against HPV 16 capsid are generally related to persistent HPV 16 infection, although a large fraction of patients with HPV 16-positive CIN fails to mount a systemic antibody response against the virus [ 107]. In transient HPV 16 infections, type-specifi c serum IgG antibodies are induced in only a minority of individuals and antibody levels wane over time [ 108]. Similarly, mucosal IgG is barely induced in transient infections. Mucosal IgA responses on the other hand, were detected more frequently and appeared to refl ect recent or ongoing HPV infection [ 109]. In established HPV infection type-specifi c antibodies may have a role in limiting the spread of active HPV infection by preventing viral reinoculation after virion release. Their role as a diagnostic marker in the process of HPV-induced disease is limited due to the lack of detectable antibody levels in a large fraction of persistently infected individuals.
HPV vaccination
Prophylactic vaccines
the keratinocytes and thereby preventing subsequent viral entry steps. Several animal models of papil-lomavirus infection have provided convincing evidence that neutralising antibodies can prevent new infection [ 1 12, 1 13]. In humans, encouraging results have been obtained with an HPV 16 virus-like particle (VLP) vaccine. Virus-like particles consist of L 1 major capsid protein, which mimics the natural virus by self-assembly into virion conformation. In a double-blind, randomized, placebo-controlled vaccination trial an HPV 16 VLP vaccine or placebo was administered to 1533 HPV 16-negative women aged 16-23 [ 1 14]. Over the follow up period, persistent HPV 16 infections – defi ned as detection of HPV 16 DNA on two consecutive 6 monthly visits – and CIN were detected in the placebo group only. A small number of vaccine recipients was HPV 16 DNA positive at a single visit, indicating that the vaccine does not establish sterilizing immunity in all cases. However, the fact that persistent infection does not ensue, suggests that the vaccination may reduce the viral load and limit rounds of reinoculation.
The results from the initial VLP-based prophylactic vaccination trial are very promising. However, several important issues remain to be addressed, such as duration of protection – which is required to be several decades – and effi cacy in immunocompromised individuals; this in view of the high incidence of HIV in the target population in the developing world. The fact that VLPs offer genotype-specifi c protection implies that a multivalent vaccine is required, targeting the most common high-risk HPV types in order to achieve maximal protection against HPV-induced malignancy. Follow-up studies on immunized individuals may also shed light on the signifi cance of oncogenic HPV in the development of other cancers (e.g. oral and oesophageal cancer).
Therapeutic vaccines
At present, several vaccines have been tested in phase I/II clinical trials. They include peptide-based vaccines, fusion proteins, antigen-pulsed DC or recombinant vaccinia viruses (reviewed in [ 1 19, 120]). Clinical responses observed in these studies were limited to those patients with prema-lignant lesions. Modest clinical responses have been observed in patients with high-grade vulvar intraepithelial neoplasia (VIN) upon vaccination with recombinant vaccinia virus encoding the modi-fi ed oncogenes of HPV 16 and 18 or a heterologous prime-boost regimen consisting of three recombi-nant HPV 16L2E6E7 protein vaccinations followed by the recombirecombi-nant vacciniavirus vaccine [ 12 1- 123]. It must be noted, however, that these trials were not placebo controlled and therefore the naturally occurring fl uctuation of lesion size was not accounted for. Nevertheless, the initial results are encour-aging, and VIN is a candidate condition for therapeutic vaccination in view of the multifocality of the disease and limited effective treatment options. In a fraction of patients, systemic T cell responses against E6 and/or E7 associated with IFNg were detected. However, the results of the immunologi-cal analyses lacked a clear-cut correlation with cliniimmunologi-cal outcome. In a double-blind, randomised, placebo-controlled clinical trial, a plasmid-DNA based vaccine containing defi ned HPV 16/ 18 E6 and E7 encoding sequences (ZYC 10 1a) showed no signifi cant effect on the resolution (defi ned as normal or CIN I) in a population of CIN 2/3 patients. Again, no correlation was observed between HPV-specifi c immune response and clinical response [ 124].
Scope of this thesis
Previous studies of HPV-specifi c immunity have focused primarily on patients with HPV-positive lesions, who have evidently failed to control HPV infection. In this thesis, the emphasis has been shifted towards successful HPV-specifi c immunity, which is expected to be found in healthy individu-als. Issues that have been addressed include the analysis of T cell memory, the associated cytokines and the viral antigens targeted by these T cells. Besides charting successful HPV-specifi c immunity, we pinpointed several characteristics of HPV-specifi c immune failure in cervical carcinoma patients. The importance of the latter lies in the fact that the effi cacy of immunotherapeutic intervention in this patient group will strongly depend on the nature of the pre-existing HPV-specifi c immune response. As a fi rst step, we investigated HPV 16-specifi c immunity in the healthy population, which – given the common nature of HPV 16 infections – harbors a large fraction of individuals who have previously experienced HPV 16 infection. The cellular immunity against the HPV 16 early proteins E2 and E6 fre-quently observed in healthy subjects (Chapters 2 and 3) most likely represents effective HPV 16-spe-cifi c immunity induced by prior HPV 16 infection, and we compared this with the immune responses in peripheral blood of patients with HPV 16-positive (pre)malignant lesions (Chapter 4). Together with the detailed analysis of virus-specifi c CD4+ T cells derived from patients’ tumors and tumor-draining lymph nodes, this provided evidence for the existence of an anti-infl ammatory cytokine polarization of the HPV 16-specifi c T cell response in cervical carcinoma patients (Chapter 5).
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Annemieke de Jong
, Sjoerd van der Burg,
Kitty Kwappenberg, Jeanette van der Hulst,
Kees Franken, Annemieke Geluk, Krista van
Meijgaarden, Jan Wouter Drijfhout,
Gemma Kenter, Pieter Vermeij, Cornelis Melief,
Rienk Offringa
Cancer Res 62: 472-479, 2002
Abstract · The incidence of genital human papillomavirus (HPV) infections is high in young
sexu-ally active individuals. Most infections are cleared within 1 year after infection. The targets for the cellular immune response in this process of viral clearance remain to be identifi ed, but the expression pattern of the E2 protein in early infection and low-grade CIN renders this early protein a candidate antigen. We therefore studied the HPV 16 E2-specifi c T cell responses in more detail. Very strong proliferative responses against one or more peptide-epitopes derived from this antigen can be found in PBMC cultures of approximately half of the healthy donors. Further analysis revealed that at least a majority of these responses represent reactivity by memory CD4+ Th 1-type cells capable of secret-ing IFNg upon antigenic stimulation. Interestsecret-ingly, all E2-peptides against which strong responses were detected are clustered in the key functional domains of the E2-protein, which are conserved to considerable extent between HPV types. This suggests that HPV 16 E2-specifi c Th memory may be installed through encounter with HPV types other than HPV 16. Indeed, one HPV 16 E2-specifi c Th-clone was found to cross-react against homologuous peptides from other HPV types, but three other Th-clones failed to show similar cross-reactivity. Part of the HPV 16 E2-specifi c Th memory may there-fore relate to previous encounter of other HPV types, whereas the majority of the immune repertoire concerned is most likely established through infection with HPV 16 itself. Our data are the fi rst to reveal that the T cell repertoire of healthy donors can contain particularly high frequencies of E2-specifi c memory Th-cells, and suggest that boosting of this immunity can be employed for preventive and therapeutic vaccination against HPV-induced lesions.
